Crystal Structure of the “cab”-type b Class Carbonic Anhydrasefrom the Archaeon Methanobacterium thermoautotrophicum*

Received for publication, October 9, 2000, and in revised form, November 26, 2000Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009182200

Pavel Strop‡, Kerry S. Smith§, Tina M. Iverson¶, James G. Ferry§i, and Douglas C. Rees¶**

From ‡Biochemistry Option, California Institute of Technology, Pasadena, California 91125, the §Department ofBiochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, and the¶Howard Hughes Medical Institute and Division of Chemistry and Chemical Engineering, California Institute ofTechnology, Pasadena, California 91125, and the iCenter for Microbial Structural Biology, Pennsylvania State University,University Park, Pennsylvania 16802

The structure of the “cab”-type b class carbonic anhy-drase from the archaeon Methanobacterium thermoau-totrophicum (Cab) has been determined to 2.1-Å resolu-tion using the multiwavelength anomalous diffractionphasing technique. Cab exists as a dimer with a subunitfold similar to that observed in “plant”-type b class car-bonic anhydrases. The active site zinc is coordinated byprotein ligands Cys32, His87, and Cys90, with the tetrahe-dral coordination completed by a water molecule. Themajor difference between plant- and cab-type b classcarbonic anhydrases is in the organization of the hydro-phobic pocket. The structure reveals a Hepes buffer mol-ecule bound 8 Å away from the active site zinc, whichsuggests a possible proton transfer pathway from theactive site to the solvent.

Carbonic anhydrases (CAs)1 are Zn21-containing enzymesthat catalyze the reversible hydration of CO2 (1). With turnovernumbers approaching 106 s21, CAs are among the fastestknown enzymes. CAs have been found in most types of organ-isms, including mammals, plants, algae, bacteria, and archaea(2). Based on the amino acid sequences, CAs can be assigned toone of three independently evolved classes, designated a, b,and g (3).

The a class contains all mammalian CAs, as well as someCAs from algae and bacteria (3, 4). a-CAs play important rolesin respiration, secretion of HCO3

2, pH homeostasis, and ion

exchange (5, 6). Crystal structures of a-CA have revealed amonomer organized around a 10-stranded, predominantly an-tiparallel b-sheet (7–13). The catalytically active zinc is coor-dinated by three histidines and one water molecule.

g-CA has thus far been isolated and characterized only fromthe methanoarchaeon Methanosarcina thermophila (14–16),where it is proposed to facilitate the transport of CH3COO2

and to convert CO2 to HCO32 outside the cell to assist the

removal of excess CO2 generated during the growth of thisorganism on acetate. The M. thermophila g-CA exists as atrimer, with the active site located at the interface between twosubunits. Each subunit is organized around a left-handed b-he-lix that is completely distinct from the a-CA fold, although theactive site is also coordinated by three histidines, along withtwo water molecules (17, 18).

The b class includes CAs from plants, algae, bacteria, andarchaea (2, 3). In higher plants, b-CAs play an important rolein photosynthesis, by concentrating CO2 in the proximity ofribulose bisphosphate carboxylase/oxygenase for CO2 fixation(19). The purification and characterization of carbonic anhy-drase (Cab) from the thermophilic Methanobacterium thermo-autotrophicum extends this class into the archaea (20). Cab isat the phylogenetic extreme of the b class carbonic anhydrasesand forms an exclusively prokaryotic clade consisting primarilyof sequences from Gram-positive bacteria (2). In the obligatechemolithoautotroph M. thermoautotrophicum, Cab convertsCO2 to HCO3

2, suggesting that the physiological role of thisenzyme may be to provide HCO3

2 to enzymes important in CO2

fixation pathways of the microbe (21, 22).b class carbonic anhydrases can be further divided into

“plant”- and “cab”-type, based on the active site sequence con-servation (20, 23) (Fig. 1). Two crystal structures of plant-typeb-CA were recently reported from Porphyridium purpureum(P. purpureum b-CA) (24) and Pisum sativum (P. sativumb-CA) (23). The basic fold of b-CA consists of a four-stranded,parallel b-sheet core with a-helices forming right-handed cross-over connections (23, 24). The oligomerization state of b-CA isvariable, however, and P. purpureum b-CA and P. sativumb-CA exist as a dimer and octamer, respectively, although thedimer of P. purpureum b-CA resembles a tetramer, where twomonomers are fused together. In contrast to the protein liga-tion by three histidines observed in a- and g-CAs, the activesite zinc in b-CAs is coordinated by two cysteines and onehistidine, as anticipated from extended X-ray absorption finestructure spectroscopy studies (21, 25, 26). The fourth ligand isdifferent in the two b-CAs structures. In the P. sativum b-CAstructure, an acetate molecule is bound to the zinc, whereas inthe P. purpureum b-CA structure, the side chain of asparticacid (Asp151) acts as the fourth ligand. In the P. sativum b-CA

* This work was supported by National Institutes of Health GrantsGM44661 (to J. G. F) and GM45162 (to D. C. R.); a National ScienceFoundation predoctoral fellowship (to P. S.); and NASA-AMES Cooper-ative Agreement NCC2-1057 (to the Pennsylvania State UniversityAstrobiology Research Center). This work is based upon research con-ducted at the Stanford Synchrotron Radiation Laboratory, which isfunded by the Department of Energy (Office of Basic Energy Sciencesand Office of Biological and Environmental Research) and the NationalInstitutes of Health (National Center for Research Resources, NIGMS).The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

The atomic coordinates and structure factors (code 1G5C) have beendeposited in the Protein Data Bank, Research Collaboratory for Struc-tural Bioinformatics, Rutgers University, New Brunswick, NJ(http://www.rcsb.org/).

** To whom correspondence should be addressed: Howard Hughes Med-ical Institute and Division of Chemistry and Chemical Engineering, Califor-nia Institute of Technology, Mail Code 147-75CH, Pasadena, CA 91125. Tel.:626-395-8393; Fax: 626-744-9524; E-mail: dcrees@caltech.edu.

1 The abbreviations used are: CA, carbonic anhydrase; Cab, M. thermo-autotrophicum b class carbonic anhydrase; b-CA, b class carbonic anhy-drase; r.m.s., root mean square; NCS, noncrystallographic symmetry.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 13, Issue of March 30, pp. 10299–10305, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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structure, this conserved Asp interacts with a conserved Arg(Fig. 1).

Apart from the conserved zinc ligands and the Asp/Arg pair,the active site of Cab differs significantly from the plant-typeb-CAs. Cab active site residues His23, Met33, Lys53, Ala58, andVal72 are replaced in the plant-type b-CA by Gln, Ala, Phe, Val,and Tyr, respectively. Residues that are equivalent to Cabresidues Met33, Lys53, Ala58, and Val72 make up the hydropho-bic pocket in the P. sativum and P. purpureum b-CA structures.Substitutions of the two aromatic side chains by Lys and Val inCab suggest a significant redesign of the hydrophobic pocket incab-type b-carbonic anhydrase. Cab has a CO2 hydration ac-tivity with a kcat of 1.7 3 104 s21 and Km for CO2 of 2.9 mM atpH 8.5 and 25 °C (20). Cab is inhibited by iodide, nitrate, andazide; however, in contrast to plant-type b-CAs, chloride andsulfate have no effect on Cab activity. These active site substi-tutions, together with the different effects of inhibitors, implythat there might be mechanistically relevant differences in theorganization of the active sites between cab-type and plant-type enzymes. Here we present the first structure of the cab-type b-carbonic anhydrase from thermophilic methanoar-chaeon M. thermoautotrophicum, determined at 2.1-Åresolution.

EXPERIMENTAL PROCEDURES

Crystallization—Cab was overexpressed in Escherichia coli and pu-rified by a heat denaturation step followed by ion exchange chromatog-raphy as previously described (20). Crystals were grown by the hangingdrop method at 22 °C using a 5 mg/ml protein solution and a precipitantsolution containing 100 mM Hepes, pH 7.5, 35% ethanol, 12% 2-methyl-2,4-pentanediol, and 50 mM calcium acetate. The crystals belong to theorthorhombic space group P212121 with unit cell dimensions a 5 54.9 Å,b 5 113.2 Å, c 5 156.2 Å and three dimers per asymmetric unit.Crystals were transferred stepwise to mother liquor solution containing30% 2-methyl-2,4-pentanediol as a cryoprotectant and flash frozen.

Data Collection and Processing—A three-wavelength multiwave-length anomalous dispersion data set was collected at 2160 °C onbeamline 9-2 of the Stanford Synchrotron Research Laboratory with anADSC charge-coupled device detector. The fluorescence spectrum meas-ured around the zinc edge of a single crystal was used to select theinflection point (l 5 1.2832 Å), the absorption edge (l 5 1.282 Å), anda high energy remote wavelength (l 5 1.033 Å) for optimization of the

anomalous signal. All data were reduced using DENZO and scaledusing SCALEPACK (27) (Table I).

Phase Determination—The structure was determined by multiwave-length anomalous dispersion using the signal from only the intrinsiczinc atoms. The program SOLVE (28) was used to find the positions ofthe heavy atoms using the three wavelength multiwavelength anoma-lous dispersion data set using data from 20- to 2.4-Å resolution. Twozinc ions were identified and when used for phasing yielded a figure ofmerit of 0.44. Four additional zinc sites were located in an anomalousdifference Fourier map, yielding a figure of merit of 0.54. Each of the sixzinc sites corresponded to a Cab monomer, which are organized intothree tight dimers in the asymmetric unit. The initial noncrystallo-graphic symmetry (NCS) transformations were established by the re-lationships between the dimers, and the initial mask was calculatedwith the program NCSMSK (29). The program DM (30) was used forNCS averaging of the electron density maps, solvent flattening, andphase extension from 2.4- to 2.1-Å resolution. The resulting map was ofgood quality and allowed building of most of the protein.

Refinement—Alternate cycles of manual model building using theprogram O (31) and positional and individual B-factor refinement withthe program CNS (32) reduced the R and Rfree to 21.1 and 24.6%respectively, where Rfree is calculated for 5% (2875) of the reflections inthe resolution range 18–2.1 Å. The model was initially refined withstrict NCS restraints, which were released later in the refinement. Ther.m.s. deviation of bond lengths and angles are 0.013 Å and 1.7° respec-tively, with 87.9% in the most allowed region and 11.1% in the addi-tionally allowed region of the Ramachandran plot. The average B-factors are 28.6 Å2 (main chain), 32.4 Å2 (side chains), 18.6 Å2 (zincatoms), and 36.3 Å2 (solvent). An average B-factor of 30.5 Å2 is calcu-lated for all protein atoms. The final model contains 7543 proteinatoms, 3 Hepes molecules, 6 zinc atoms, 6 calcium atoms, and 409 watermolecules, for a total of 8015 atoms (Table II).

RESULTS AND DISCUSSION

Structural Organization of Cab—The overall fold of the Cabmonomer (Fig. 2A) consists of a four-stranded parallel b-sheetcore with strand order 2-1-3-4. Monomers in each dimer arerelated by a 2-fold axis centered between strands b2. Theoverall dimensions of the dimer are ;40 3 45 3 50 Å (Fig. 2B).Three dimers, designated AB, CD, and EF, are present in theasymmetric unit. In the structurally conserved b-sheet region(residues 26–32, 52–57, 80–88, 149–157, and 163–167), ther.m.s. deviations in Ca positions between the dimers average0.24 Å. Using Ca positions for residues 24–170, the correspond-

FIG. 1. Alignment of b-CA sequences. Cab, M. thermoautotrophicum; PPnterm, P. purpureum N-terminal domain; PPcterm, P. purpureumC-terminal domain; P.S., P. sativum; ECcynT, E. coli. Zinc ligands are colored yellow, the conserved Asp/Arg pair is red, and residuesdifferentiating cab-type and plant-type are blue.

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ing r.m.s. deviations are 0.34 Å between dimers AB and CD, 1.0Å between dimers AB and EF, and 1.1 Å between dimers CDand EF. The relatively large r.m.s. deviations for the latter twopairs of dimers reflect the poor ordering for residues 91–126 inmonomer E. Excluding these residues, the r.m.s. deviationsdrop to ;0.28 Å. The r.m.s. deviation in the b-sheet regionbetween the A and B, C and D, and E and F subunits of the Cabare ;0.6 Å, while the r.m.s. deviation for all Ca between thesubunits are ;0.8 Å.

The regions with the largest conformational variation in-clude the N-terminal residues 1–23 (involved in crystal pack-ing), residues 92–95, and residues 120–125. While the N-ter-minal residues 1–23 are well defined in monomers A, C, and E,residues 13–23 are disordered in monomers B, D, and F. Res-idues 92–95 and 120–125 form a hinge region for helices a4 anda5 and are well ordered in monomers B and D, slightly disor-dered in monomers A, C, and F, and disordered in monomer E.The variability in region 90–125 between the six crystallo-graphically independent monomers suggests that these resi-dues are conformationally mobile. Overall, with the exceptionof the noted regions, the three dimers in the asymmetric unitare very similar. Unless stated otherwise, only dimer AB willbe used in future discussions.

Six calcium ions were located on the surface of Cab and atthe crystal packing interfaces between Cab dimers. Most likely,these calcium ions do not play any structural or catalytical roleand are a result of the crystallization conditions that contained50 mM calcium acetate.

Cab Oligomerization—b-CAs have been found in differentoligomeric states ranging from dimers (Oryza sativa, P. purpu-reum) to tetramers (E. coli) to octamers (P. sativum) (20, 23, 24,

33). Although analytical ultracentrifugation results suggestthat Cab exists as a homotetramer (21), Cab appears to form adimer in the crystal. There are numerous interactions stabiliz-ing the dimer involving residues in b-strand b2 and helices a2,a3, a4, and a5. Hydrogen bonds between residues 56 and 57from strand b2 in both subunits result in a formation of a10-stranded b sheet. Helices a4 and a5 extend out and makeextensive contacts with the other monomer in the dimer (Fig.2B). The interface area between these two subunits was foundto be ;2110 Å2/subunit, which is ;21% of the total (;10,000Å2) monomer accessible surface area as calculated with GRASP(34). Residues A2–A23 pack against a symmetry-related mole-cule burying 860 Å2, resulting in the formation of a continuousribbon through the crystal (Fig. 3). Residues B2–B12 mostlikely fold back and pack against the B monomer in the sameway that A2–A12 packs against the symmetry-related mole-cule. Residues B13–B23 have no visible electron density andare disordered. A similar type of crystal packing forming acontinuous ribbon has been observed in the sterile a motifdomain of the human EphB2 receptor and, together with otherevidence, was proposed to be functionally important (35). Thecrystal packing interaction seen in Cab can also be described asa linear (open-ended) domain swapped oligomer, where theswapped domain consists of a 12-residue a-helix (36, 37). It isunclear whether residues 1–24 could facilitate the formation ofa higher oligomerization state under physiological conditionsor whether residues A2–A23 also fold back and pack againstmonomer A. Based on modeling considerations, although theN-terminal region is highly flexible, formation of a Cab tet-ramer similar to the one seen in the P. purpureum structure isunlikely in the absence of conformational changes due to stericclashes of helices a4 and a5. Other contacts between Cabdimers in the crystal are relatively small (#540 Å2) and, basedon their complementarity and size (38, 39), are also unlikely tosupport formation of stable tetramers.

Comparison of Cab and P. sativum and P. purpureum Struc-tures—The fold of Cab is similar to that of the P. sativum andP. purpureum b-CAs (Fig. 4). While the b-sheet core is con-served, significant secondary structure differences are evident,mostly in the regions at the N terminus, at the C terminus, andin the region containing residues 90–125. The N termini of theP. sativum and P. purpureum b-CA structures extend out,forming a long helix that packs against a second monomer,making additional dimer interactions. In Cab, the N terminusis involved in crystal packing and does not adopt the sameconformation observed in the P. sativum and P. purpureumb-CA structures. Cab is one of the smallest b-CAs known andlacks an extended C terminus. In the P. sativum b-CA struc-ture, the C terminus forms a long b-strand that mediates

TABLE IData collection statistics

Numbers in parentheses indicate values for highest resolution bin (2.38–2.30 Å for l1, 2.49–2.4 Å for l2, and 2.17–2.10 Å for l3. cen, centric;acen, acentric; anom, anomalous.

Data collection Peak l1 Inflection l2 Remote l3

Wavelength (Å) 1.282 1.2832 1.0333Resolution range (Å) 20–2.3 20–2.4 20–2.1Unique observations 43,450 38,663 57,495Total observations 156,889 139,023 208,917Completeness (%) 97.9/(92.0) 98.5/(96.5) 98.8/(98.2)Rsym

a 5.9 (21.8) 5.1 (16.7) 6.3 (24.3)^I/s& 24 (7.0) 25 (8.6) 21 (5.7)Rcullis (cen/acen/anom)b 0.79/0.78/0.86 0.74/0.76/0.91 NA/NA/0.92c

Phasing power (cen/acen)d 0.81/1.26 0.94/1.38Figure of merit to 2.4-Å resolution (cen/acen) 0.52/0.57

a Rsym 5 ¥uIobs 2 Iavgu/¥Iobs.b Rcullis 5 lack of closure error/iso-ano difference.c NA, not applicable.d Phasing power 5 r.m.s. heavy atom structure factor/r.m.s. lack of closure error.

TABLE IIRefinement statistics

Parameter Value

Space group P212121Cell dimensions

a (Å) 54.67b (Å) 113.21c (Å) 156.18Resolution (Å) 20–2.1Rcryst (%)a 21.1Rfree (%)b 24.6

r.m.s. deviationsBond lengths (Å) 0.014Bond angles (degrees) 1.70Torsion angles (degrees) 22.9Improper torsion angles (degrees) 1.39

a Rcryst 5 S u Fo 2 Fcu/Su Fou.b Rfree 5 Rcryst calculated for 5% of reflections omitted from the

refinement.

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octamerization. In Cab, residues 90–125 form two helices (a4,a5) that project out to cover the second monomer (Fig. 2B) andfold back to start helix a6. In both the P. sativum and P.purpureum b-CA structures, this segment is longer, formsthree helices instead of two, and folds back earlier to create twoadditional turns of helix a6. In the central b-sheet region, ther.m.s. deviations in Ca positions between Cab and P. sativumand P. purpureum structures are 0.62 and 0.56 Å, respectively.

Active Site—The active site cleft is located at the C terminusof the parallel b-sheet and is largely sequestered from solvent.Each Cab subunit contains one zinc atom that resides at theinterface of the two monomers (Fig. 2B), although the coordi-nation residues (Cys32, Cys90, and His87) originate from thesame monomer. One water molecule completes the tetrahedralcoordination sphere of the zinc. Although the crystallizationconditions contained 50 mM calcium acetate, no acetate wasfound in the active site, unlike in the P. sativum b-CA structure

(Fig. 5A). The average coordination distances from the sixactive sites are as follows: Cys32 Sg-Zn (2.42 6 0.03 Å), His87

Ne-Zn (2.11 6 0.04 Å), Cys90 Sg-Zn (2.40 6 0.04 Å), and H2O-Zn(2.15 6 0.06 Å). The four zinc ligands form a number of hydro-gen bonds with the surrounding residues. His87 hydrogen-bonds with the carbonyl oxygen of residue Thr88; Cys32 Sghydrogen-bonds with the amide nitrogens of residues Lys22,Asp34, and Gly59; Cys90 hydrogen-bonds with the amide nitro-gen of residue Met92; and the coordinating water moleculemakes a hydrogen bond to Asp34. Arg36 forms the conservedAsp/Arg pair with Asp34 and also interacts with Asp89 and awater molecule.

Rather unexpectedly, the two active sites A and B in the Cabdimer exhibit significant differences, which are reflected in ther.m.s. deviations between monomers in a dimer (;0.65 Å) beingconsistently larger than between the equivalent monomers indifferent dimers such as A and C, or B and D (0.24 Å). The

FIG. 2. Structure of Cab. A, ribbon diagram of the A monomer of Cab. The color changes from blue (N terminus) to red (C terminus). B, dimerof Cab. Monomer A is shown in magenta, monomer B is shown in blue, and zinc atoms are shown in gray. Monomer A is shown in the sameorientation for both A and B. Hepes bound to subunit A is shown in a ball-and-stick representation. The images were created with BOBSCRIPT(45) and RASTER3D (46).

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active sites in monomers A, C, and E have a Hepes buffermolecule bound near the active site. The sulfate group of theHepes is located ;8 Å from the zinc atom, and the sulfateoxygens hydrogen bond with Lys53 Nz, Ser35 Og, and the amidenitrogen of Ser35 (Fig. 5B). The equivalent to Ser35 is present inboth plant-type b-CAs and in Cab, while Lys53 is unique to thecab-type b-CAs. Asp34, which makes a hydrogen bond to thezinc-coordinating water molecule, is also within hydrogenbonding distance (;3.0 Å) to the Hepes sulfate group. Anotherdifference between the two active sites is the conformation ofresidues 13–24. In the active site of subunit A, residues B13–B24 are disordered and have no visible electron density. In theactive site of subunit B, however, residues A13–A24 are welldefined and extend out to participate in crystal packing. Su-perposition of residues in the active sites of subunits A and B(Fig. 5B) indicates that the Hepes molecule would stericallyclash with residues Arg16 and Asp17 if residues 13–24 adoptedthe same conformation as in active site B.

Comparison of P. sativum b-CA and Cab Active Sites—Thezinc-coordinating residues (Cys32, His87, and Cys90) of Cab andP. sativum b-CA superimpose closely (Fig. 6A). The water mol-ecule serving as the fourth zinc ligand in Cab adopts a positionsimilar to the O1 of the acetate ligand in the P. sativum b-CAstructure. The conserved residue Asp34 is held in place byforming two hydrogen bonds to Arg36, as do the corresponding

residues Asp162 and Arg164 in the P. sativum b-CA. The follow-ing five active site substitutions distinguish the cab-type andplant-type b-carbonic anhydrases: H23Q, M33A, K53F, A58V,and V72Y. The superposition of Cab and P. sativum b-CAstructures clearly shows the distinctions in active site organi-zation by these residues. His23, found on a flexible loop, is ;25Å away from the catalytic zinc and probably is not a part of theactive site. On the other hand, the equivalent residue Gln151 inthe P. sativum b-CA structure occupies a position similar to theHepes sulfate group that is ;8 Å away from the zinc atom andis possibly involved in ligand binding (23). The hydrophobicpocket of Cab is quite different from that of plant-type b-CA(Fig. 6A). In P. sativum b-CA structure, the hydrophobic pocketis formed by Phe179, Val184, and Tyr205. The correspondingresidues in Cab (Lys53, Ala58, and Val72) constitute a more openand less hydrophobic pocket.

Comparison of P. purpureum b-CA and Cab Active Sites—Again, the protein ligands to the zinc in the two structuressuperpose closely (Fig. 6B). The fourth ligand is different, sincethe side chain of Asp151 in the P. purpureum b-CA structurecoordinates the zinc instead of the water molecule seen in Cabstructure. Asp151 of P. purpureum b-CA is equivalent to Asp34

of Cab. As a consequence of the zinc coordination by Asp151 inthe P. purpureum b-CA structure, this residue cannot pair withthe conserved Arg, and Arg153 has flipped away from the active

FIG. 4. Stereo diagram showing the superposition of a backbone trace of Cab (yellow), P. sativum b-CA (green), and P. purpureumb-CA (blue). The main differences are at the N terminus, C terminus, and in the a4, a5 region.

FIG. 3. Stereo view of the crystalpacking of Cab showing the continu-ous ribbon created by packing of he-lix a1 against a crystallographicallyrelated molecule. Monomer A is in ma-genta, with helix a1 in green. Monomer Bis in blue, with helix a1 in red.

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FIG. 6. Stereo diagram showing the superposition of the active site of Cab, P. sativum, and P. purpureum b-CAs including zinc,zinc-coordinating residues, and the conserved residues differentiating between cab- and plant-type b-CA. A, Cab is shown in yellow;P. sativum b-CA is shown in green. The coordinating water molecule in Cab is shown in red. B, Cab is shown in yellow; P. purpureum b-CA is shownin blue. The coordinating water molecule in Cab is shown in red.

FIG. 5. Stereo view of the zinc envi-ronment in Cab. A, the simulated an-nealing (?Fo? 2 ?Fc?) omit map, calculatedafter omitting the four ligands and thezinc, is contoured at 4s (blue) and 15s(red). A simulated annealing omit mapcalculated with only the coordinating wa-ter molecule omitted is shown contouredat 4s (green). B, Hepes binding to theactive site of subunit A. Hepes is in pur-ple, with hydrogen bonds shown as dottedlines. The loop shown in green wouldsterically clash with Hepes if Hepesbound to the active site of subunit B as itis observed to bind to the active site ofsubunit A.

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site. The adjacent Ser152 has moved by ;4 Å and adopts adifferent conformation in the P. purpureum b-CA structure(Fig. 6B). The hydrophobic pocket arrangement of P. purpu-reum b-CA is very similar to that of P. sativum b-CA, and thedifferences between the cab-type and plant-type hydrophobicpocket have already been discussed.

Mechanism—The catalytic mechanism of carbonic anhy-drases has been most extensively studied for the a-CA class (5,6, 40–42). The zinc hydroxide mechanism established for thisclass provides an appropriate framework for discussing thecatalytic mechanism of Cab. In the first part of the CO2 hydra-tion reaction, CO2 binds in the hydrophobic pocket and proba-bly interacts with the amide nitrogen of Thr199. This threonineis known as the “gatekeeper,” and the side chain plays animportant role in the a-CAs, together with Glu106, in orientingthe CO2 molecule for attack by the zinc-bound hydroxide. In theP. sativum b-CA structure, Asp162, Gly224, and Gln151 arethought to play the same role in orienting CO2 for this attack(23). In Cab, Asp34 and Gly91 are in the same orientation asAsp162 and Gly224 in P. sativum b-CA structure and might alsohelp to orient CO2. His23, the equivalent of P. sativum b-CAGln151, is, however, disordered in active sites A, C, and E, andin active sites B, D, and F it is at the beginning of a segment ofresidues that pack against the symmetry-related molecule andlies 25 Å away from the active site zinc.

The second, rate-limiting, step in the CO2 hydration reactioninvolves the regeneration of a hydroxide ion from the zinc-bound water molecule. In a-CAII, the zinc ion is located in adeep funnel and requires a proton shuttle to transfer the protonto the bulk solvent. His64 of a-CAII adopts multiple conforma-tions, which facilitates accepting the proton from the zinc-bound water molecule and delivering it to buffer in bulk solu-tion (43). In g-CA, Glu84 exhibits multiple conformations andhas been proposed to participate in a proton shuttle (18, 44).Residues with multiple conformations have not been describedin the active site of any b-CA structure determined so far. Sincethe b-CA active site is closer to the surface of the protein thanthe a-CA active site, a protein-mediated proton shuttle mightnot be necessary. The b-CA reaction rate depends on bufferconcentration, implying that proton transfer can be rate-limit-ing under certain conditions (21). In the Cab structure, a Hepesbuffer molecule was found near the active sites A, C, and E. TheHepes sulfate group is located ;8 Å away from the zinc atomand lies within hydrogen bonding distance of Asp34, whichmakes a hydrogen bond to the zinc bound water molecule. Inthe P. purpureum b-CA structure, the equivalent of Asp34 actsas the fourth zinc ligand, and in the proposed mechanism itplays a role in the proton transfer (24). Therefore, the mostplausible pathway for proton transfer in Cab is from the zinc-bound water molecule to Asp34 and then to the sulfate group ofthe bound Hepes molecule or a solvent molecule. The confor-mation of residues 1–25 in active sites B, D, and F is incom-patible with Hepes binding, and residues 13–25 must adoptdifferent conformations for Hepes to bind (Fig. 5B). The mobil-ity of residues 1–25 and 92–125 might allow buffer molecules todiffuse into the active site and serve as the proton acceptornecessary to regenerate the zinc-bound hydroxide.

Acknowledgments—We thank Jessica Wuu and Radu Georgescu forhelp with crystallizations and Brian R. Crane and Alex M. Bilwes forhelp with data collection.

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Structure of “cab”-type b Class Carbonic Anhydrase 10305

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Pavel Strop, Kerry S. Smith, Tina M. Iverson, James G. Ferry and Douglas C. Rees Methanobacterium thermoautotrophicum

Class Carbonic Anhydrase from the ArchaeonβCrystal Structure of the ''cab''-type

doi: 10.1074/jbc.M009182200 originally published online November 28, 20002001, 276:10299-10305.J. Biol. Chem. 

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